The principal objective of this proposal is to use T4 lysozyme as a model system to better understand the factors that determine the folding, stability, structure and function of proteins. The specific research to be accomplished includes the following: (1) The body of structural, thermodynamic, and kinetic information on mutant T4 lysozymes will be used, both within the group and in collaboration with others, to test and improve computer-assisted protein design. (2) An attempt will be made to simplify the protein folding problem by identifying which residues, or combination of residues, are critical for the folding and stability of T4 lysozyme. We want to understand, not only how given residues contribute to stability, but also the relative importance of local versus non-local effects in protein folding. (3) Cavities within T4 lysozyme will be exploited to better understand protein-ligand interaction and to optimize rational approaches to drug selection and docking (in collaboration with B. Shoichet). (4) We will attempt to engineer a long-distance, ligand-induced conformational switch in a protein. (5) Neutron crystallography will be used to characterize catalytic intermediates and to compare dynamical processes in crystals of T4 lysozyme with those in solution. (6) The contribution of solvent to protein folding and stability will be further analyzed. (7) The effects of pressure on proteins with cavities will be determined.